Type 2 diabetes is a huge a growing health problem in the United States today and is caused by impaired insulin signaling (i.e. decreased insulin action), primarily in skeletal muscle. Insulin binding to its receptor initiates a series of phosphorylation events that cause glucose transport vesicles (GLUT-4) that reside inside the cell to translocate to the surface and facilitate glucose uptake. Defects at any of steps in insulin signaling can impair GLUT-4 translocation and lead to insulin resistance. Skeletal muscle is unique in that in addition to insulin signaling, exercise (i.e. repeating muscle contractions) also promotes skeletal muscle glucose uptake. This effect is independent of insulin and provides an alternate means of entry for glucose into skeletal muscle that is insulin resistant. A vast amount of research has demonstrated that these 2 pathways are distinct, to a point, but converge at the protein AS160 and the newly identified homologue, TBC1D1. Animal studies demonstrate that both insulin and exercise increase phosphorylation of AS160 and TBC1D1. However, due to lack of phosphosite-specific antibodies, there is little information available on the site-specific regulation of these proteins by insulin or exercise in humans. Our laboratory has circumvented the need for phosphosite-specific antibodies when studying the phosphorylation of other important proteins (e.g. IRS-1) by using HPLC- electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). These mass spectrometry techniques can be adapted and applied to signaling proteins such as AS160 and TBC1D1 to better understand their post-translational regulation in vivo. Therefore, the overall objective of this proposal is three fold: 1. to identify known and novel phosphorylation sites on AS160 and TBC1D1 in human skeletal muscle using mass spectrometry based techniques 2. to assess the regulation of these phosphorylation sites by insulin and exercise and 3. to assess the effect of insulin resistance in altering site-specific phosphorylation of AS160 and TBC1D1. The long-term scientific objective is utilize the information gained from this study to better understand how insulin and exercise promote skeletal muscle glucose uptake and to find defects in insulin-resistant skeletal muscle that can be targeted by pharmaceuticals or exercise to reverse insulin resistance. PUBLIC HEALTH RELEVANCE: Decreasing the incidence of type 2 diabetes requires a full understanding of the underlying cause of this disease (i.e. diminished insulin action). This study will increase our understanding of how insulin and exercise affect signaling proteins that regulate glucose uptake and how these signals may be altered in insulin resistant individuals. This information may help us to target important regulation sites and improve insulin action.